title: Expression and structural characterization of anti-T-antigen single-chain antibodies (scFvs) and analysis of their binding to T-antigen by surface plasmon resonance and NMR spectroscopy.
abstract: T-antigen (Galβ1-3GalNAcα-1-Ser/Thr), also known as Thomsen-Friedenreich antigen (TF antigen), is an oncofetal antigen commonly found in cancerous tissues. Availability of anti-T-antigen human antibodies could lead to the development of cancer diagnostics and therapeutics. Four groups of single-chain variable fragment (scFv) genes were previously isolated from a phage library (Matsumoto-Takasaki et al. (2009) Isolation and characterization of anti-T-antigen single chain antibodies from a phage library. BioSci Trends 3:87-95.). Here, four anti-T-antigen scFv genes belonging to Group 1-4 were expressed and produced in a Drosophila S2 cell expression system. ELISA and surface plasmon resonance (SPR) analyses confirmed the binding activity of 1E8 scFv protein to various T-antigen presenting conjugates. NMR experiments provided evidence of the folded nature of the 1E8 scFv protein. ScFv-ligand contact was identified by STD NMR, indicating that the galactose unit of T-antigen at the non-reducing end was primarily recognized by 1E8 scFv. This thus provides direct evidence of T-antigen specificity.
authors.affiliation: Department of Applied Biochemistry, Tokai University School of Engineering, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan; and Structural Glycobiology Team, Systems Glycobiology Research Group, RIKEN-Max Planck Joint Research Center, RIKEN Global Research Cluster, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Yuasa
authors.forename: Tsubasa
authors.initials: T
authors.lastname: Koyama
authors.forename: Ganesh P
authors.initials: GP
authors.lastname: Subedi
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Yamaguchi
authors.forename: Misao
authors.initials: M
authors.lastname: Matsushita
authors.forename: Yoko
authors.initials: Y
authors.lastname: Fujita-Yamaguchi
mesh_terms: D000818:Animals; D000957:Antigens, Viral, Tumor / Q000276:immunology*; D002460:Cell Line; D004330:Drosophila; D015870:Gene Expression; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D020349:Surface Plasmon Resonance
chemical_list: D000957:Antigens, Viral, Tumor; D057127:Single-Chain Antibodies
keywords: Drosophila S2 cell expression; T-antigen; anti-carbohydrate antibodies; insect cell; phage display